Degumming is an important step in oil and fat refining. Traditional degumming by hydration is of high economic cost, high consumption of materials and energy and serious environmental pollution. As such, many works were devoted to apply a degumming by enzyme in the degumming step of oil and fat refining. As compared to the traditional method, the method of degumming by enzyme has great advantages in environmental protection, economic effect, and quality, etc., as it could improve economic benefits, reduce energy consumption and emission and reduce the ecological environment pollution. One kind of enzymes used in degumming of oil and fat is phospholipase. Phospholipase C (PLC) exhibits significant advantages in, such as increasing yield of diacylglycerols (DAG) and reducing loss of the produced oil, as compared to the other enzymes for degumming.
Phospholipase C specific to phosphatidylcholine from Bacillus cereus (BC-PC-PLC) is a phospholipase C that was studied early. BC-PC-PLC consists of 283 amino acids in its full length, with a signal peptide of 24 amino acids and a leader peptide of 14 amino acids. The mature peptide consists of 245 amino acids. See, such as, Johansen, T., Holm, T., Guddal, P. H., Sletten, K., Haugli, F. B., Little, C.(1988), “Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus ” Gene 65(2):293-304).
The crystal structure of BC-PC-PLC have been reported, which consists of many helix domains and at least three Zn2+ binding sites, with the catalytic site at the aspartic acid at position 55. See, such as, Hough., E., Hansen, L. K., Birknes, B., Jynge, K., Hansen, S., Hordvik, A., Little, C., Dodson, E., Derewenda, Z. (1989) “High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus.” Nature. 338:357-60. Heterogenous expression of BC-PC-PLC was less studied. It has been reported that BC-PC-PLC was expressed in Bacillus subtilis and pichia pastoris. See, for example, Durban, M. A., Silbersack, J., Schweder, T., Schauer, F., Bornscheuer, U. T. (2007) High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis. Appl Microbiol Biotechnol 74(3):634-639; and Seo, K. H, Rhee J. I. (2004) High-level expression of recombinant phospholipase C from Bacillus cereus in Pichia pastoris and its characterization. Biotechnol Lett 26(19):1475-1479.
Modified phospholipase C having, such as, a higher enzymatic activity, is desired in the art.